Role of Hypertonic Saline and Pentoxifylline on Neutrophil Activation and Tumor Necrosis Factor-α Synthesis: A Novel Resuscitation Strategy

Abstract

Hypertonic saline (HS) and pentoxifylline (PTX) have been shown to modulate polymorphonuclear neutrophil (PMN) functions after shock and sepsis. We hypothesized that a combination of HS and PTX (HSPTX) would down-regulate PMN functions and inflammatory mediator synthesis more effectively than each alone, possibly by acting at different steps of the signaling pathways, ultimately leading to an enhanced effect. Whole blood from healthy volunteers was stimulated with lipopolysaccharide (LPS) (100 μg/mL), f-methionyl-leucyl-phenylalanine (1 μmol/L), and phorbol 12-myristate 13-acetate (1 μg/mL). Baseline oxidative burst was measured by flow cytometry. Two different concentrations of NaCl to achieve increases of 10 mmol/L (HS10) and 40 mmol/L (HS40) above isotonicity, simulating increases in sodium levels seen after infusion of 3% HS and 7.5% HS, were used. PTX (2 mmol/L), HS10, HS40, HSPTX10, and HSPTX40 were added to whole blood concomitantly to the activators. PMN CD14 and CD11b expression were measured by flow cytometry and tumor necrosis factor-α levels by enzyme-linked immunosorbent assay in LPS-stimulated whole blood. The combination of PTX with HS10 and with HS40 markedly decreased LPS (27 ± 7 and 23 ± 6 vs. 100; p < 0.01)-, f-methionyl-leucyl-phenylalanine (54 ± 11 and 55 ± 8 vs. 100; p < 0.05)–, and phorbol 12-myristate 13-acetate (30 ± 4 and 54 ± 9 vs. 100; p < 0.01 and p < 0.05, respectively)– induced PMN oxidative burst. Furthermore, a significant decrease in LPS-induced neutrophil CD11b expression after PTX treatment (79 ± 5 vs. 100; p < 0.05) and HSPTX40 (68 ± 7 vs. 100; p < 0.05) was observed. HSPTX10 (7 ± 0.6; p < 0.001) or HSPTX40 (6 ± 1.4; p < 0.001) markedly decreased tumor necrosis factor-α production to levels similar to those observed with PTX alone (6.5 ± 0.5; p < 0.001) and significantly lower than HS10 (110 ± 4.9; p < 0.001) and HS40 alone (83 ± 1.5; p < 0.001) HSPTX down-regulates neutrophil activation and proinflammatory mediator synthesis more effectively that HS alone. HSPTX may have significant applications as a novel fluid resuscitation strategy in hemorrhagic shock.